Identification and Rescue of -Synuclein Toxicity in
Parkinson Patient-Derived Neurons
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Chung, Chee Yeun, Vikram Khurana, Pavan K. Auluck, Daniel F.
Tardiff, Joseph R. Mazzulli, Frank Soldner, Valeriya Baru, et al.
“Identification and Rescue of -Synuclein Toxicity in Parkinson
Patient-Derived Neurons.” Science 342, no. 6161 (October 24,
2013): 983–987.
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http://dx.doi.org/10.1126/science.1245296
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American Association for the Advancement of Science (AAAS)
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Author's final manuscript
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Thu May 26 21:31:48 EDT 2016
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http://hdl.handle.net/1721.1/92886
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Published as: Science. 2013 November 22; 342(6161): 983–987.
HHMI Author Manuscript
Identification and Rescue of α-Synuclein Toxicity in Parkinson
Patient-Derived Neurons
Chee Yeun Chung1,†, Vikram Khurana1,2,†, Pavan K. Auluck1,3, Daniel F. Tardiff1, Joseph R.
Mazzulli2, Frank Soldner1, Valeriya Baru1,4, Yali Lou1,4, Yelena Freyzon1, Sukhee Cho5,
Alison E. Mungenast5, Julien Muffat1, Maisam Mitalipova1, Michael D Pluth6, Nathan T.
Jui6, Birgitt Schüle7, Stephen J. Lippard6, Li-Huei Tsai5,8, Dimitri Krainc2, Stephen L.
Buchwald6, Rudolf Jaenisch1,8, and Susan Lindquist1,4,9,*
1Whitehead
Institute for Biomedical Research, Cambridge, MA 02142, USA
2Department
of Neurology, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA
HHMI Author Manuscript
3Department
of Pathology (Neuropathology), Massachusetts General Hospital and Harvard
Medical School, Boston, MA 02114, USA
4Howard
Hughes Medical Institute, Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA
5The
Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
6Department
7The
of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Parkinson’s Institute, Sunnyvale, CA 94085
8Howard
Hughes Medical Institute, Cambridge, MA
9Department
of Biology, Massachusetts Institute of Technology, Cambridge, MA
HHMI Author Manuscript
Abstract
The induced pluripotent stem (iPS) cell field promises a new era for in vitro disease modeling.
However, identifying innate cellular pathologies, particularly for age-related neurodegenerative
*
Correspondence to: Dr Susan Lindquist, lindquist_admin@wi.mit.edu.
†These authors contributed equally to this work and are listed alphabetically.
Supplementary Materials
www.sciencemag.org
Materials and Methods
Figures S1–S10
Table S1
References (29–40) [Note: The numbers refer to any additional references cited only within the Supplementary Materials]
Author contributions: CYC, VK and SL conceptualized the study, designed the experiments, and wrote the paper. VK developed the
human IPS cell-derived cortical synucleinopathy model, assisted by YL. Pluripotent cell lines, advice on experimental design or
technical expertise were provided by FS, JRM, JM, MM and RJ. FS reprogrammed the WIBR-IPS-SYNTRPL line from fibroblasts
provided by BS. CYC developed the rat cortical synucleinopathy model, assisted by LB. The mKate2-tagged constructs were
generated by YF. CYC and VK performed all experiments except: fig. 1D and E, fig. 2E, fig. S5A and B (PKA); fig. 4A (DFT); fig.
4C, fig. S6C (JRM/DK); fig. S2, H–I (AEM/SC/LT); fig. S1 (YF/YL). The small molecule NAB2 was synthesized by NTJ and SLB,
based on a yeast screen performed by DFT. FL2 dye synthesis and technical advice were provided by MDP and SJL. CYC, VK and
SL are inventors on a pending patent application related to work described in this paper.
Chung et al.
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diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved
proteotoxic mechanisms from yeast to human to discover and reverse phenotypic responses to αSynuclein (αSyn), a key protein involved in Parkinson’s disease (PD). We generated cortical
neurons from iPS cells of patients harboring αSyn mutations, who are at high risk of developing
PD dementia. Genetic modifiers from unbiased screens in a yeast model of αSyn toxicity led to
identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress,
accumulation of ER-associated degradation (ERAD) substrates and ER stress. A small molecule
identified in a yeast screen, and the ubiquitin ligase Nedd4 it activates, reversed pathologic
phenotypes in these neurons.
HHMI Author Manuscript
Neurodegenerative dementias are devastating and incurable diseases for which we
desperately need tractable cellular models to investigate pathologies and discover
therapeutics. Parkinson disease dementia (PDD) is a major debilitating non-motor
manifestation of Parkinson’s disease, affecting as many as 80% of patients (1). The best
pathologic correlate of PDD is neuron loss and pathologic aggregation of αSyn within deep
layers of the cerebral cortex (1). Contursi kindred patients, who harbor an autosomal
dominant and highly penetrant A53T mutation in αSyn, manifest prominent PD and
dementia (2, 3). iPS cells from a female member of this kindred (Table S1) have recently
been mutation-corrected to control for genetic background effects (4). To establish a model
for cortical synucleinopathy, we differentiated two pairs of subclones from these lines (fig.
S1) into cortical neurons (fig. S2 and S3).
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Over 12 weeks of differentiation, cultures consisted primarily of excitatory glutamatergic
neurons mixed with glia (fig. S2, C to E; fig. S3). To identify neurons, neural precursors
were infected prior to differentiation with lentiviruses expressing enhanced yellow
fluorescent protein (eYFP) or red fluorescent protein (RFP) under the control of the synapsin
promoter (fig. S2G). When co-cultured, A53T and corrected neurons were electrically active
at 8 weeks of differentiation. They exhibited similar calcium fluxes and electrophysiology
(fig. S2, H and I). The majority of neurons were immunopositive for Tbr1, a transcription
factor indicating developing deep cortical layers (fig. S2E) (5). αSyn was robustly
expressed, but only after neuronal differentiation (fig. S2, C to F). αSyn was both
cytoplasmic and punctate in neuronal processes (fig. S2, C to E). Thus, these cells provide a
relevant substrate for examining early αSyn-related cortical pathologies.
It has been difficult to establish neurodegenerative phenotypes in iPS cell-derived neurons
that can be solely attributable to disease-causing genetic mutations. Previous studies
accelerated degenerative phenotypes with toxins such as oxidative stressors (6–8). In
addition, inconsistent differentiation precludes these cells from being used in highthroughput screening. To address these problems, we turned to a yeast platform in which
αSyn-expression results in toxicity (9, 10) and disease-relevant phenotypes, including focal
accumulation of αSyn, mitochondrial dysfunction, αSyn-mediated vesicle trafficking
defects, links to genetic and environmental risk factors, and sensitivity to αSyn dosage (9–
12). We reasoned that unbiased genetic analysis in this system could guide discovery of
innate pathologic phenotypes.
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Previous unbiased yeast genetic screens, encompassing 85% of the yeast proteome,
identified robust modifiers of αSyn toxicity (9, 12). We first tested Fzf1, a transcriptional
regulator of nitrosative stress responses (13) that suppressed αSyn toxicity in yeast (fig. 1A).
Nitrosative stress is caused by nitric oxide (NO) and related redox forms. Though it is not
known if there is a direct causal connection between nitrosative stress and αSyn toxicity, the
nitration of tyrosine residues is increased in postmortem brain from synucleinopathy patients
(14, 15).
To determine if nitrative damage occurs to yeast proteins in direct response to αSyn, we
took advantage of an antibody to nitrotyrosine. In yeast, this antibody exhibited minimal
background in control strains, allowing us to detect intense protein nitration that was tightly
dependent on αSyn dosage (fig. 1B). Importantly, nitration was a highly specific response to
αSyn toxicity and was not observed with other neurodegenerative disease proteins expressed
at equally toxic levels, including Abeta peptide, TDP-43, polyQ-expanded huntingtin and
Fus (fig. 1B).
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Fzf1 expression strongly decreased protein nitration induced by αSyn (fig. 1C). Next, we
asked if αSyn toxicity could be tuned by altering the production of NO. In yeast, NO levels
are regulated by switching between distinct isoforms of mitochondrial cytochrome c oxidase
(COX5): deletion of COX5A increases NO; deletion of COX5B decreases it (16). Indeed,
these manipulations increased and decreased nitrotyrosine levels in response to αSyn (fig.
1D). Toxicity increased and decreased commensurately (fig. 1E). Thus, in yeast, nitrosative
stress is not simply a consequence of αSyn toxicity, but contributes to toxicity.
To investigate a connection between αSyn and nitrosative stress in neurons we employed
FL2, a copper and fluorescein-based NO sensor (17). We optimized the use of FL2 with rat
primary cortical cultures (fig. S4), a neuronal syucleinopathy model. αSyn overexpression
increased the FL2 signal, with a perinuclear distribution in the cell body (fig. 2A) that
partially co-localized with the endoplasmic reticulum (ER; fig. 2B). High density of
processes and mixed cell types hindered intensity measurements outside well-defined
neuronal cell bodies.
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Having optimized the FL2 assay in rat neurons, we turned to our Parkinson patient-derived
cortical neurons at 8 weeks of differentiation. Two isogenic pairs of A53T and mutationcorrected neurons were differentiated in parallel and labeled with synapsin-RFP (see fig.
S2G). Intraneuronal FL2 signals increased in A53T neurons relative to corrected neurons,
again most readily visualized in the cell body. As in rodent neurons, there was partial colocalization of this signal with an ER marker (fig. 2C). Cytoplasmic nitrotyrosine staining
also accumulated in mutant neurons compared to corrected neurons (fig. 2D). Similarly,
cytoplasmic nitrotyrosine staining was prominent in neurons and neuropil of post-mortem
frontal cortex from another subject in the same kindred (18), but not in control brain (fig.
2E).
The yeast synucleinopathy model exhibits ER stress, ER-associated degradation (ERAD)
substrate accumulation and defective trafficking from the ER to Golgi (12). ER stress has
also been described in a mouse syucleinopathy model (19). Because NO was visualized in
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the vicinity of the ER in neurons (fig. 2), we asked whether modulating NO levels modulates
ER stress. Indeed, manipulating COX5 isoforms, to increase and decrease NO levels,
commensurately altered the unfolded protein response (fig. S5A) and the ER accumulation
of carboxypeptidase Y (CPY; S5B), a well-characterized ERAD substrate that traffics
between the ER and vacuole (12). This required the presence of αSyn (fig. S5), implying a
connection between nitrosative and ER stress in the context of αSyn toxicity.
Correspondingly, two hallmarks of ER stress – PDI (protein disulfide isomerase) and BIP
(binding immunoglobulin protein) – increased at 12 weeks of differentiation in the A53T
neurons compared to corrected cells. Levels of CHOP (CCAAT enhancer-binding protein
homologous protein), a component of ER stress-induced apoptosis, did not change,
indicating cellular pathology was still at an early-stage (fig S5C).
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Next, we assessed the accumulation and trafficking of three ERAD substrates implicated in
neurodegeneration: Glucocerebrosidase (GCase), Neuroserpin and Nicastrin (20). GCase
mutations are common risk factors for PD and confer risk for cognitive impairment in this
disease (21). GCase accumulates in the ER of cultured cells over-expressing αSyn (22). ER
forms of GCase and Nicastrin accumulated, and the ratio of post ER/ER forms declined in
A53T compared to mutation-corrected patient neurons starting at four weeks (fig. 3, A and
B; fig. S, 6A to C). Neuroserpin was not affected at the timepoints we examined (fig. 3, A
and B; fig. S6, A to C). Levels of neuron-specific markers were unaffected (fig. 3C and fig.
S6C). These findings were consistent in multiple rounds of differentiation, robust to distinct
differentiation protocols (fig. S6C). Phenotypes were not present in the undifferentiated iPS
cell lines (fig. S6D). Thus, ERAD dysfunction is an early and progressive cellular phenotype
in response to mutated α-Syn in patient neurons.
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The increase in the ER form of GCase, and the decrease in the post-ER to ER ratio, was
recapitulated in the brain of an A53T patient (fig. 3C and fig. S7B). Cortex from sporadic
PD samples exhibited the same trend (fig. S7). We also analyzed cortical neurons generated
from the iPS cells of a male patient of the “Iowa kindred”. This patient harbored a
triplication of the wild-type αSyn gene and manifested aggressive dementia in addition to
parkinsonism (Table S1). Aged cortical neurons generated from a male human embryonic
stem cell line BG01(23) served as a control. ERAD substrates accumulated (fig. 3A and fig.
S6B) and ER stress increased (fig. S5C) in neurons from this patient, closely phenocopying
A53T cells.
Another suppressor of αSyn toxicity recovered in the yeast screen was Hrd1 (fig. 3D, left)
(9). Hrd1 is a highly conserved E3 ubiquitin ligase (Synoviolin-1 or Syvn1 in humans) that
plays a critical role in ERAD from yeast to human. In primary rat cortical neurons lentiviral
expression of Syvn1 rescued αSyn toxicity in a dose-dependent manner (fig. 3D, right).
Syvn1 also reduced nicastrin and GCase accumulation in the ER of the A53T patient cortical
neurons (fig. 3E).
Next, we tested the ability of NAB2 (24) to rescue the pathological phenotypes we
discovered here in both yeast cells and PD neurons. NAB2, an N-arylbenzimidazole, was
recovered in a yeast screen of more than 180,000 small molecules and rescues αSyn toxicity
in yeast by activating the Rsp5/Nedd4 pathway (24). This protein is another highly
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conserved ubiquitin ligase, and plays a key role in regulating vesicle trafficking (25, 26).
NAB2 reduced protein nitration in the yeast syucleinopathy model (fig. 4A) and decreased
NO levels in A53T patient neurons (fig. 4B). Moreover, NAB2 reduced the accumulation of
immature ER forms of CPY in yeast (fig. 4A). It increased the post-ER forms, and decreased
the immature forms, of Nicastrin and GCase in PD patient neurons (fig. 4C and fig. S8).
Furthermore, NAB2 analogs that were inactive in yeast (24) were also inactive in human
neurons (fig. S9). Connecting NAB2 back to the ubiquitin ligase, we used a lentivirus to
overexpress Nedd4. This phenocopied the effects of the compound, increasing the mature
forms of Nicastrin and GCase (fig. 4D).
Conserved biology in a cross-species cellular discovery platform, as described here enabled
the discovery of innate pathologic phenotypes in neurons derived from patients with PD. It
also enabled the identification of genes and small molecules that reverted these phenotypes
(24) (fig. S10). A similar approach might be useful in the study of other PD-relevant
phenotypes identified in yeast, including mitochondrial dysfunction and perturbed metal ion
homeostasis (9,11). The existence of other yeast models of neurodegenerative diseases
suggest that this approach will be generalizable to other diseases (11, 27, 28).
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Dennis Dickson, Larry Golbe and John Trojanowki for postmorterm tissue or data; David Pincus for the
UPR reporter; Jan Pruszak, Patti Wisniewski, Iain Cheeseman for giving important technical advice; Raaji
Alagappan, Tenzin Lungiangwa and Ping Xu for superb technical assistance; Sandro Santagata, Luke Whitesell,
Mel Feany, Dirk Landgraf and Linda Clayton for fruitful discussion and critical comments on the manuscript. Grant
support was provided by a Howard Hughes Medical Institute Collaborative Innovation Award (SL), JPB
Foundation grants (SL), NIH/NIA K01 AG038546 (CYC), American Brain Foundation and Parkinson’s Disease
Foundation Clinician-Scientist Development Award (VK), NIH 5 R01CA084198 (RJ), JBP foundation (LHT) and
NSF (SJL).
References
HHMI Author Manuscript
1. Irwin DJ, et al. Neuropathologic substrates of Parkinson disease dementia. Ann Neurol. 2012;
72:587–598. [PubMed: 23037886]
2. Spira PJ, Sharpe DM, Halliday G, Cavanagh J, Nicholson GA. Clinical and pathological features of
a Parkinsonian syndrome in a family with an Ala53Thr alpha-synuclein mutation. Ann Neurol.
2001; 49:313–319. [PubMed: 11261505]
3. Markopoulou K, et al. Clinical, neuropathological and genotypic variability in SNCA A53T familial
Parkinson’s disease. Variability in familial Parkinson’s disease. Acta Neuropathol. 2008; 116:25–
35. [PubMed: 18389263]
4. Soldner F, et al. Generation of isogenic pluripotent stem cells differing exclusively at two early
onset Parkinson point mutations. Cell. 2011; 146:318–331. [PubMed: 21757228]
5. Saito T, et al. Neocortical Layer Formation of Human Developing Brains and Lissencephalies:
Consideration of Layer-Specific Marker Expression. Cerebral Cortex. 2011; 21:588–596. [PubMed:
20624841]
6. Nguyen HN, et al. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to
oxidative stress. Cell stem cell. 2011; 8:267–280. [PubMed: 21362567]
Science. Author manuscript; available in PMC 2014 November 22.
Chung et al.
Page 6
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author Manuscript
7. Byers B, et al. SNCA Triplication Parkinson’s Patient’s iPSC-derived DA Neurons Accumulate αSynuclein and Are Susceptible to Oxidative Stress. PLoS ONE. 2011; 6:e26159. [PubMed:
22110584]
8. Cooper O, et al. Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from
patients with familial Parkinson’s disease. Sci Transl Med. 2012; 4:141ra90.
9. Gitler AD, et al. Alpha-synuclein is part of a diverse and highly conserved interaction network that
includes PARK9 and manganese toxicity. Nat Genet. 2009; 41:308–315. [PubMed: 19182805]
10. Outeiro TF, Lindquist S. Yeast cells provide insight into alpha-synuclein biology and
pathobiology. Science. 2003; 302:1772–1775. [PubMed: 14657500]
11. Khurana V, Lindquist S. Modelling neurodegeneration in Saccharomyces cerevisiae: why cook
with baker’s yeast? Nat Rev Neurosci. 2010; 11:436–449. [PubMed: 20424620]
12. Cooper AA, et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in
Parkinson’s models. Science. 2006; 313:324–328. [PubMed: 16794039]
13. Sarver A, DeRisi J. Fzf1p regulates an inducible response to nitrosative stress in Saccharomyces
cerevisiae. Mol Biol Cell. 2005; 16:4781–4791. [PubMed: 16014606]
14. Giasson BI, et al. Oxidative damage linked to neurodegeneration by selective alpha-synuclein
nitration in synucleinopathy lesions. Science. 2000; 290:985–989. [PubMed: 11062131]
15. Gómez-Tortosa E, et al. Patterns of protein nitration in dementia with Lewy bodies and
striatonigral degeneration. Acta Neuropathol. 2002; 103:495–500. [PubMed: 11935266]
16. Castello PR, et al. Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on
its nitric oxide production and on hypoxic signaling. Proc Natl Acad Sci USA. 2008; 105:8203–
8208. [PubMed: 18388202]
17. Pluth MD, Tomat E, Lippard SJ. Biochemistry of Mobile Zinc and Nitric Oxide Revealed by
Fluorescent Sensors. Annu Rev Biochem. 2011; 80:333–355. [PubMed: 21675918]
18. Kotzbauer PT, et al. Fibrillization of alpha-synuclein and tau in familial Parkinson’s disease caused
by the A53T alpha-synuclein mutation. Exp Neurol. 2004; 187:279–288. [PubMed: 15144854]
19. Colla E, et al. Endoplasmic reticulum stress is important for the manifestations of αsynucleinopathy in vivo. J Neurosci. 2012; 32:3306–3320. [PubMed: 22399753]
20. Tabuchi K, Chen G, Südhof TC, Shen J. Conditional forebrain inactivation of nicastrin causes
progressive memory impairment and age-related neurodegeneration. J Neurosci. 2009; 29:7290–
7301. [PubMed: 19494151]
21. Alcalay RN, et al. Cognitive performance of GBA mutation carriers with early-onset PD: the
CORE-PD study. Neurology. 2012; 78:1434–1440. [PubMed: 22442429]
22. Mazzulli JR, et al. Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional
pathogenic loop in synucleinopathies. Cell. 2011; 146:37–52. [PubMed: 21700325]
23. Mitalipova M, et al. Human embryonic stem cell lines derived from discarded embryos. STEM
CELLS. 2003; 21:521–526. [PubMed: 12968106]
24. Tardiff DF, et al. Phenotypic screening and chemical genetics reveals a “druggable” Rsp5/Nedd4
network that ameliorates alpha-synuclein toxicity. Science (Co-submission with this work).
25. Haynes CM, Caldwell S, Cooper AA. An HRD/DER-independent ER quality control mechanism
involves Rsp5p-dependent ubiquitination and ER-Golgi transport. J Cell Biol. 2002; 158:91–101.
[PubMed: 12105183]
26. Donovan P, Poronnik P. Nedd4 and Nedd4-2: Ubiquitin ligases at work in the neuron. Int J
Biochem Cell Biol. 2012:1–5.
27. Elden AC, et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with
increased risk for ALS. Nature. 2010; 466:1069–1075. [PubMed: 20740007]
28. Treusch S, et al. Functional links between Aβ toxicity, endocytic trafficking, and Alzheimer’s
disease risk factors in yeast. Science. 2011; 334:1241–1245. [PubMed: 22033521]
29. Golbe LI, et al. Clinical genetic analysis of Parkinson’s disease in the contursi kindred. Ann
Neurol. 1996; 40:767–775. [PubMed: 8957018]
30. Muenter MD, et al. Hereditary form of parkinsonism--dementia. Ann Neurol. 1998; 43:768–781.
[PubMed: 9629847]
Science. Author manuscript; available in PMC 2014 November 22.
Chung et al.
Page 7
HHMI Author Manuscript
31. Singleton AB, et al. alpha-Synuclein locus triplication causes Parkinson’s disease. Science. 2003;
302:841. [PubMed: 14593171]
32. Waters CH, Miller CA. Autosomal dominant Lewy body parkinsonism in a four-generation family.
Ann Neurol. 1994; 35:59–64. [PubMed: 8285594]
33. Yeger-Lotem E, et al. Bridging high-throughput genetic and transcriptional data reveals cellular
responses to alpha-synuclein toxicity. Nat Genet. 2009; 41:316–323. [PubMed: 19234470]
34. Soldner F, et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral
reprogramming factors. Cell. 2009; 136:964–977. [PubMed: 19269371]
35. Kim J-E, et al. Investigating synapse formation and function using human pluripotent stem cellderived neurons. 2011:1–6.
36. Chambers SM, et al. Highly efficient neural conversion of human ES and iPS cells by dual
inhibition of SMAD signaling. Nat Biotechnol. 2009; 27:275–280. [PubMed: 19252484]
37. Shi Y, Kirwan P, Livesey FJ. Directed differentiation of human pluripotent stem cells to cerebral
cortex neurons and neural networks. Nat Protoc. 2012; 7:1836–1846. [PubMed: 22976355]
38. Lim MH. Preparation of a copper-based fluorescent probe for nitric oxide and its use in
mammalian cultured cells. Nat Protoc. 2007; 2:408–415. [PubMed: 17406602]
39. McQuade LE, et al. Visualization of nitric oxide production in the mouse main olfactory bulb by a
cell-trappable copper(II) fluorescent probe. Proc Natl Acad Sci USA. 2010; 107:8525–8530.
[PubMed: 20413724]
40. Pincus D, et al. BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the
Unfolded Protein Response. PLoS Biol. 2010; 8:e1000415. [PubMed: 20625545]
HHMI Author Manuscript
HHMI Author Manuscript
Science. Author manuscript; available in PMC 2014 November 22.
Chung et al.
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HHMI Author Manuscript
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Fig. 1.
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A specific link between αSyn toxicity and nitrosative stress is identified in yeast (A) Fzf1
overexpression reduces αSyn toxicity in yeast (IntTox) measured by growth of serially
diluted yeast. (B) Protein nitration levels were measured by immunublotting for 3nitrotyrosine (3-NT). Strains expressing low (NoTox), intermediate (IntTox) and high
(HiTox) levels of αSyn were analyzed (11). Neurodegeneration-related models with
equivalent toxicity (expressing Aβ [β–amyloid peptide], Htt72Q [Huntingtin exon 1 with 72
glutamines] or Fus) were not similarly affected. (C) Fzf1 expression reduced αSyn-induced
increase in nitration. (D and E) NO-increasing deletion of Cox5A (ΔCox5A) increased
protein nitration levels, whereas the NO-decreasing Cox5B deletion (ΔCox5B) reduced
protein nitration levels (D). Toxicity was determined by propidium iodide stained cells using
flow cytometry (E). Data represented as mean ± SEM, ***; p<0.001 One way ANOVA with
Bonferroni post-hoc test.
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Fig. 2.
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Nitrosative stress is implicated in rat and human iPS neuron synucleinopathy models, and in
the brain of a patient harboring the A53T αSyn mutation. (A) Primary rat cortical cultures
were infected with AAV2 mKate2 or A53T αSyn-mKate2 (synapsin promoter). Cells were
loaded with FL2 and live-imaged using a confocal microscope (neuronal soma: perforated
circle). Perinuclear FL2 signal partially co-localized with ER tracker in rat neurons. (B–C)
Increased NO (FL2) and 3-nitrotyrosine (3-NT) levels in human αSynA53T iPS neurons at 8
weeks. For the FL2 experiment (B), neural progenitors were transduced with lentivirus-RFP
(synapsin promoter) to mark neurons. (D) Postmortem frontal cortex from a patient
harboring A53T mutation exhibited increased 3-NT immunoreactivity. All data represented
as mean ± SEM, *; p<0.05, ***; p<0.001, two tail t-test.
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Fig. 3.
Accumulation of ERAD substrates in patient cortical neurons is reversed by Synoviolin. (A
and B) Cortical iPS neurons from αSynA53T (A1, A2), αSyncorrected (C1, C2), a male
human ES line (BGO1) and αSyntriplication (S3) patients were harvested at 8–12 weeks. ER
or post ER forms were distinguished based on sensitivity to Endoglycosidase H (Endo H) in
GCase (glucocerebrosidase), Nicastrin and Nrspn (neuroserpin) (n=4~6, two tail t-test
compared to control samples at each time point). (C) ER forms of GCase also accumulated
in postmortem cortex from the A53T patient. (D) Co-expression of Hrd1 or its mammalian
homolog, Synoviolin, reduced αSyn toxicity in yeast and rat cortical neurons. Rat cultures
were transduced by lentivirus encoding Synoviolin with varying multiplicity of infection
(MOI) and co-transduced with lenti-αSynA53T or lenti-LacZ. Cellular ATP content was
measured (One way ANOVA with Bonferroni post-hoc test). (E) Lentiviral transduction of
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Synoviolin reduced accumulation of ER forms of GCase and Nicastrin in αSynA53T iPS
cortical neurons at 8–12 weeks. Baseline PD levels were equated to % control established in
fig. 3 to depict biological significance of the change. All data represented as mean ± SEM,
*; p<0.05, **; p<0.01; ***; p<0.001).
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Fig. 4.
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A small molecule modifier identified in an unbiased yeast screen and its target correct
analogous defects in yeast and patient neurons. (A) NAB2 ameliorates αSyn-induced ER
accumulation of CPY and nitrosative stress in the yeast model. (B) NAB2 (5 μM) decreases
nitric oxide (FL2) levels in αSynA53T iPS neurons labeled with Synapsin-RFP. (C) NAB2
increases post-ER forms of and ameliorates the ER accumulation of GCase and Nicastrin in
αSynA53T iPS neurons. (D) Lentiviral delivery of Nedd4 phenocopies the NAB2 treatment,
increasing mature forms of GCase and Nicastrin. Baseline PD levels were equated to
%control established in fig. 3. All data represented as mean ± SEM (*; p<0.05, **; p<0.01,
***; p<0.001, two tail t-test compared to control condition).
Science. Author manuscript; available in PMC 2014 November 22.